This research focuses on studying the role of tyrosine phosphorylation in the mechanism of insulin action at the cellular and molecular level. Over the past 5 years with the support of this grant, we have defined many of the structural features of the receptor required for kinase activity, identified and purified IRS-1 (the major insulin receptor substrate in most cells), cloned this protein at the cDNA and genomic levels in three species, identified at least one of the down-stream signals generated by IRS-1, developed reconstitution systems to validate the role of the receptor and its substrate in insulin action and characterized alterations in this signal transduction system in physiologic and pathologic states. A total of 68 original reports and 14 reviews and chapters are published, in press or submitted as a result of this grant. The major goal over the next 5 years will be to determine the role of IRS-1 in insulin action and to continue to explore other potential pathways by which this tyrosine kinase acts. We will study the role of IRS-1 in coupling the insulin receptor tyrosine kinase to down-stream effector systems in insulin action. Three systems will be developed and used: (1) Xenopus oocytes have IGF-1 receptors, but lack endogenous IRS-1. IRS-1 will be expressed directly in the oocyte or in a baculovirus system and microinjected into oocytes where coupling to down-stream actions can be studied. The effect of co-injection of antibodies, peptides, or other proteins can also directly be evaluated. (2) In vitro mutagenesis and expression of IRS-1 and related signaling molecules will be performed in CHO and 3T3-F442A cells. (3) Analysis of IRS-1 function in intact animals by targeted gene knock-out by homologous recombination. We will characterize the regulation of IRS-1 in vivo and in vitro at the translational and post- translational levels in both intact animals and cells in culture with particular emphasis on pathways of non-lysosomal degradation. The effects of hormonal treatment and disease will be studied. We will also determine the nature of the coupling of the insulin receptor tyrosine phosphorylation cascade to down-stream effectors of insulin action such as PI 3, MAP and S6 kinases, possible interacting and redundant pathways and especially how the nucleus is linked to the cytoplasmic signal. Finally, we will attempt to identify other effector systems involved in insulin action which might interact directly with the insulin receptor by coprecipitation assays and expression screening.